US11419931B2 - Compositions comprising bacterial strains - Google Patents

Compositions comprising bacterial strains Download PDF

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US11419931B2
US11419931B2 US17/024,628 US202017024628A US11419931B2 US 11419931 B2 US11419931 B2 US 11419931B2 US 202017024628 A US202017024628 A US 202017024628A US 11419931 B2 US11419931 B2 US 11419931B2
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cells
compositions
seq
strain
mrx0518
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US20210169950A1 (en
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Domenico PANZICA
Amy Beth HOLT
Suaad AHMED
Anna ETTORRE
Imke Elisabeth MULDER
Philip Cowie
Emma RAFTIS
Emma Elizabeth Clare HENNESSY
Delphine Louise Claudette Laute-Caly
Aurélie Pascale Patricia COUTURIER-MAILLARD
Margaret Inkster DELDAY
Marsilio ADRIANI
Maria CHRISTOFI
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4D Pharma Research Ltd
Armistice Capital Master Fund Ltd
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Priority claimed from GBGB1804384.4A external-priority patent/GB201804384D0/en
Priority claimed from GBGB1809953.1A external-priority patent/GB201809953D0/en
Priority claimed from GBGB1811900.8A external-priority patent/GB201811900D0/en
Priority claimed from GBGB1812378.6A external-priority patent/GB201812378D0/en
Priority claimed from GBGB1813444.5A external-priority patent/GB201813444D0/en
Priority claimed from GBGB1813423.9A external-priority patent/GB201813423D0/en
Priority claimed from GBGB1816834.4A external-priority patent/GB201816834D0/en
Priority claimed from GBGB1817641.2A external-priority patent/GB201817641D0/en
Priority claimed from GBGB1901199.8A external-priority patent/GB201901199D0/en
Priority claimed from GBGB1901218.6A external-priority patent/GB201901218D0/en
Priority claimed from GBGB1901993.4A external-priority patent/GB201901993D0/en
Priority claimed from GBGB1901992.6A external-priority patent/GB201901992D0/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is in the field of compositions comprising bacterial strains isolated from the mammalian digestive tract and the use of such compositions in the treatment of disease, in particular in stimulating the immune system in the treatment of disease.
  • the human intestine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1].
  • the human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Finnicutes [2].
  • the successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions.
  • the enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host.
  • the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-4].
  • Streptococcus and Veillonella strains and to a lesser extent, Enterococcus and Lactobaccillus strains have been suggested to have immunomodulatory effects, with varying effects on different cytokines in vitro, suggesting that data obtained in vitro with individual strains are unlikely to adequately represent immune responses to mixtures of gut microbiota communities in vivo [88].
  • the relationship between different diseases and different bacterial strains, and the precise effects of particular bacterial strains on the gut and at a systemic level and on any particular types of diseases are poorly characterised.
  • the inventors have developed new compositions comprising a bacterial strain of the species Enterococcus gallinarum that can be used in stimulating the immune system and treating and preventing disease.
  • the inventors have identified that strains of the species Enterococcus gallinarum can potently activate the immune system and can treat cancer, which indicates that they may able to also treat other diseases where activation of the immune system may be useful.
  • the invention therefore provides a composition comprising a bacterial strain of the species Enterococcus gallinarum , for use in stimulating the immune system in subject.
  • the invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum , for use in treating, preventing or delaying immunosenescence.
  • the invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum , for use as a vaccine adjuvant.
  • the invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum , for use in enhancing a cell therapy, such as CAR-T.
  • the bacteria used in the invention is the strain deposited under accession number 42488 at NCIMB.
  • the bacteria used in the invention is the strain deposited under accession number 42761 at NCIMB.
  • FIG. 1A Mouse model of breast cancer—changes in tumour volume post tumour induction and a table indicating the statistical significance between each two treatments at each time point.
  • FIG. 1C Mouse model of breast cancer—infiltrating immune cells. Scatter plots represent cell counts of different immune markers from individual animals from each treatment group.
  • FIG. 1D Mouse model of breast cancer—Cytokine production in tumour lysates. Columns represent the mean pg/mL of total protein from each treatment group. *p ⁇ 0.05 between groups using one-way ANOVA followed by Dunnett's multiple comparisons test.
  • FIG. 1E Mouse model of breast cancer—Cytokine production in blood plasma. Columns represent the mean pg/mL from each treatment group (+/ ⁇ SEM).
  • FIG. 1F Representative images of ileum cryosections from vehicle, MRx0518 and anti-CTLA-4-treated mice immuno-labelled with antibodies against CD8a (lower panels) and counter-stained with DAPI (upper panels).
  • FIG. 1G Plot quantifying animal study subsets with more than 3 CD8 ⁇ + cells per field taken from the ileum crypt region of mice treated with vehicle, MRx0518 or anti-CTLA-4.
  • FIG. 2 Mouse model of lung cancer—changes in tumour volume post tumour induction and a table indicating the statistical significance between each two treatments at each time point.
  • FIG. 3A Mouse model of liver cancer—liver weight.
  • FIG. 3B Mouse model of kidney cancer—changes in tumour volume post tumour induction and a table indicating the statistical significance between each two treatments at each time point.
  • FIG. 4A Cytokine levels (pg/mLmL) in immature dendritic cells (No bacteria).
  • FIG. 4B Cytokine levels (pg/mLmL) in immature dendritic cells after the addition of LPS.
  • FIG. 4C Cytokine levels (pg/mLmL) in immature dendritic cells after the addition of MRx0518.
  • FIG. 4D Cytokine levels (pg/mLmL) in immature dendritic cells after the addition of MRx0518 and LPS.
  • FIG. 5A Cytokine levels in THP-1 cells (No bacteria).
  • FIG. 5B Cytokine levels in THP-1 cells after addition of bacterial sediment.
  • FIG. 5C Cytokine levels in THP-1 cells after the addition of MRx0518MRx0518 alone or in combination with LPS.
  • FIG. 6 Immunostimulatory response—TNF ⁇
  • FIG. 7 Immunostimulatory response—TNF ⁇
  • FIG. 8 Immunostimulatory response—IL-12p70
  • FIG. 9 Immunomodulatory response—IL-10
  • FIG. 10 Immunostimulatory response—IL-8
  • FIG. 11 Immunostimulatory response—IL-23
  • FIG. 12 Immunostimulatory response—IL-1 ⁇
  • FIG. 13 Immunostimulatory response—IL-6
  • FIG. 14 Mechanism of action—activation of NF ⁇ B
  • FIG. 15 Mechanism of action—activation of TLR5
  • FIG. 16A A schematic representation of the treatment schedule of the different groups used in Example 8 described herein below.
  • FIG. 16B Mean tumour volume in mice bearing a tumour formed by EMT-6 cells.
  • the mice were either untreated or treated with a YCFA vehicle (Vehicle), MRx0518 bacteria in YCFA medium (MRx0518), an anti-CTLA-4 antibody and YCFA medium (Anti-CTLA-4) or a combination of MRx0518 and the anti-CTLA-4 antibody.
  • the provided table indicates the statistical significance between each two treatments at each time point.
  • FIG. 17 Mouse model of breast cancer—tumour volume.
  • FIG. 18 API 50 CHL profile of MRx0554.
  • FIG. 19 Mechanism of action—TLR9 activation by MRx0518 (MRx0518 LV ), heat-killed MRx0518 (MRx0518 HK ) and MRx0518 culture supernatant (MRx0518 SN ) in HEK-BlueTM hTLR9 reporter cell lines.
  • ODN2006 was used as a positive control and YCFA medium was included as a negative control for MRx0518 SN .
  • the bar graph represents an average of at least three biological replicates. Statistical analysis was performed using GraphPad Prism (ordinary one-way ANOVA analysis followed by Tukey's Multiple comparison test). Statistically significant differences with the relevant control are shown on the graphs as ****(p ⁇ 0.0001).
  • FIGS. 21A-21D In-vitro cytokine production by ( FIG. 21A ) PBMC cells; ( FIG. 21B ) Splenocytes; or ( FIG. 21C ) THP-1 cells; which were treated with YCFA+medium (“Vehicle”) or cell-free bacterial supernatant of MRx0518 (“MRx0518”).
  • FIG. 21D shows fold change in cytokine expression following treatment of CaCo-2 cells with live bacteria (“MRx0518”) relative to untreated cells.
  • Cells were either untreated (“Untreated”), treated with YCFA blank media (“10% YCFA”) or treated with MRx0518 cell-free bacterial supernatant (“10% MRx0518”).
  • FIGS. 22A-22D NF- ⁇ B promoter activation in ( FIG. 22A ) HEK-BlueTM-hNOD2 cells; ( FIG. 22B ) HEK-BlueTM-hTLR4 cells; ( FIG. 22C ) HEK-BlueTM-hTLR9 cells or ( FIG. 22D ) HEK-BlueTM-hTLR5 cells.
  • Cells were either untreated, treated with YCFA medium (“YCFA”), treated with MRx0518 (“MRx0518”) or treated with positive controls.
  • YCFA YCFA
  • MRx0518 treated with MRx0518
  • FIG. 23 Heat map representing NanoString analysis of EMT6 tumour microenvironment following treatment with YCFA vehicle (“Vehicle”) or MRx0518 (“MRx0518”).
  • compositions of the invention comprise a bacterial strain of the species Enterococcus gallinarum .
  • the examples demonstrate that bacteria of this genus are useful for stimulating the immune system and for treating disease.
  • Enterococcus gallinarum forms coccoid cells, mostly in pairs or short chains. It is motile and colonies on blood agar or nutrient agar are circular and smooth. Enterococcus gallinarum reacts with Lancefield group D antisera.
  • GenBank accession number for a 16S rRNA gene sequence of Enterococcus gallinarum is AF039900 (disclosed herein as SEQ ID NO:1). An exemplary Enterococcus gallinarum strain is described in [11].
  • strain MRx0518 The Enterococcus gallinarum bacterium deposited under accession number NCIMB 42488 was tested in the Examples and is also referred to herein as strain MRx0518. References to MRx0518 and MRx0518 are used interchangeably. A 16S rRNA sequence for the MRx0518 strain that was tested is provided in SEQ ID NO:2. Strain MRx0518 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 16 Nov. 2015 as “ Enterococcus sp” and was assigned accession number NCIMB 42488.
  • strain MRx0518 comprises a chromosome and plasmid.
  • a chromosome sequence for strain MRx0518 is provided in SEQ ID NO:3 OF WO2017/085520.
  • a plasmid sequence for strain MRx0518 is provided in SEQ ID NO:4 OF WO2017/085520. These sequences were generated using the PacBio RS II platform.
  • strain MRx0554 The Enterococcus gallinarum bacterium deposited under accession number NCIMB 42761 was also tested in the Examples and is also referred to herein as strain MRx0554. References to MRx0554 and MRx0554 are used interchangeably. Strain MRx0554 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 22 May 2017 as “ Enterococcus gallinarum MRx0554” and was assigned accession number NCIMB 42761. The genome sequence of this bacterium is disclosed herein as SEQ ID NO:2 OF WO2018/215782.
  • Ns in the sequence represent gaps between the contigs.
  • N may represent an A, G, C or T nucleotide.
  • a 16S rRNA gene sequence for the MRx0554 strain is provided in SEQ ID NO:3.
  • SEQ ID NO:3 represents the full length sequence present in the assembly, rather than a consensus of the five 16S genes present in MRx0554.
  • the bacterial strain for use in the invention has a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 2.
  • the sequence identity is to SEQ ID NO:2.
  • the bacterial strain for use in the invention has the 16s rRNA gene sequence represented by SEQ ID NO:2.
  • the bacterial strain for use in the invention has a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:3.
  • Bacterial strains that are biotypes of the bacterium deposited under accession number 42488 are also expected to be effective for stimulating the immune system.
  • a biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.
  • Strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 42488.
  • substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome).
  • a biotype strain has at least 98% sequence identity across at least 98% of its genome or at least 99% sequence identity across 99% of its genome.
  • Biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG) 5 (SEQ ID NO: 4), or REP or [12].
  • Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42488.
  • a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0518 deposited as NCIMB 42488 and comprises a 16S rRNA gene sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ ID NO:2.
  • a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0518 deposited as NCIMB 42488 and has the 16S rRNA sequence of SEQ ID NO:2.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO2017/085520.
  • the bacterial strain for use in the invention has a chromosome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:3 OF WO2017/085520 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:3 OF WO2017/085520.
  • the bacterial strain for use in the invention may have a chromosome with at least 90% sequence identity to SEQ ID NO:3 OF WO2017/085520 across 70% of SEQ ID NO:3 OF WO2017/085520, or at least 90% sequence identity to SEQ ID NO:3 OF WO2017/085520 across 80% of SEQ ID NO:3 OF WO2017/085520, or at least 90% sequence identity to SEQ ID NO:3 OF WO2017/085520 across 90% of SEQ ID NO:3 OF WO2017/085520, or at least 90% sequence identity to SEQ ID NO:3 OF WO2017/085520 across 100% of SEQ ID NO:3 OF WO2017/085520, or at least 95% sequence identity to SEQ ID NO:3 OF WO2017/085520 across 70% of SEQ ID NO:3 OF WO2017/085520, or at least 95% sequence identity to SEQ ID NO:3 OF WO2017/085520 across 80% of SEQ ID NO:3 OF WO2017/085520
  • the bacterial strain for use in the invention has a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520.
  • the bacterial strain for use in the invention has a plasmid with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:4 OF WO2017/085520 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:4 OF WO2017/085520.
  • the bacterial strain for use in the invention may have a plasmid with at least 90% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 70% of SEQ ID NO:4 OF WO2017/085520, or at least 90% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520, or at least 90% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 90% of SEQ ID NO:4 OF WO2017/085520, or at least 90% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 100% of SEQ ID NO:4 OF WO2017/085520, or at least 95% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 70% of SEQ ID NO:4 OF WO2017/085520, or at least 95% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO2017/085520 and a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO2017/085520, for example as described above, and a 16S rRNA sequence with sequence identity to any of SEQ ID NO:1 or 2, for example as described above, preferably with a 16s rRNA sequence that is at least 99% identical to SEQ ID NO: 2, more preferably which comprises the 16S rRNA sequence of SEQ ID NO:2, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520, as described above.
  • the bacterial strain for use in invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO2017/085520, for example as described above, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520, as described above, and is effective for stimulating the immune system.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO2017/085520, for example as described above, and a 16S rRNA sequence with sequence identity to any of SEQ ID NOs: 1 or 2, for example as described above, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520, as described above, and is effective for stimulating the immune system.
  • the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ ID NO: 2 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2) and a chromosome with at least 95% sequence identity to SEQ ID NO:3 OF WO2017/085520 across at least 90% of SEQ ID NO:3 OF WO2017/085520, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520, as described above, and which is effective for stimulating the immune system.
  • SEQ ID NO: 2 for example, which comprises the 16S rRNA sequence of SEQ ID NO:2
  • a chromosome with at least 95% sequence identity to SEQ ID NO:3 OF WO2017/085520 across at least 90% of SEQ ID NO:3 OF WO2017/085520 and optionally comprises a plasmid with sequence identity to SEQ ID
  • the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA gene sequence represented by SEQ ID NO: 2 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2) and a chromosome with at least 98% sequence identity (e.g. at least 99% or at least 99.5% sequence identity) to SEQ ID NO:3 OF WO2017/085520 across at least 98% (e.g.
  • SEQ ID NO:3 OF WO2017/085520 across at least 99% or at least 99.5%) of SEQ ID NO:3 OF WO2017/085520, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520, as described above, and which is effective for stimulating the immune system.
  • the bacterial strain for use in the invention is a Enterococcus gallinarum and has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ ID NO: 2 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2) and a chromosome with at least 98% sequence identity (e.g. at least 99% or at least 99.5% sequence identity) to SEQ ID NO:3 OF WO2017/085520 across at least 98% (e.g.
  • SEQ ID NO:3 OF WO2017/085520 across at least 99% or at least 99.5%) of SEQ ID NO:3 OF WO2017/085520, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520, as described above, and which is effective for stimulating the immune system.
  • strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42488 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing.
  • FAFLP fluorescent amplified fragment length polymorphism
  • rep repetitive DNA element
  • protein profiling or partial 16S or 23s rDNA sequencing.
  • such techniques may be used to identify other Enterococcus gallinarum strains.
  • strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 42488 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example, [13]).
  • ARDRA amplified ribosomal DNA restriction analysis
  • biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42488.
  • the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMérieux).
  • the bacterial strain used in the invention is:
  • Enterococcus gallinarum strains that are useful in the compositions and methods of the invention, such as biotypes of the bacterium deposited under accession number NCIMB 42488, may be identified using any appropriate method or strategy, including the assays described in the examples. For instance, strains for use in the invention may be identified by assessing their effects on cytokine levels, as performed in the examples. In particular, bacterial strains that have similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 42488 may be useful in the invention. A useful strain will have comparable immune modulatory activity to the NCIMB 42488 strain.
  • a biotype strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
  • a biotype strain that may be used in the invention is a strain which is able to elicit comparable effects on the cancer disease models shown in the Examples when administered in the method of the invention.
  • the bacterial strain used in the invention is:
  • the bacterial strain used in the invention is:
  • the bacterial strain used in the invention is an extracellular ATP producer, for example one which produces 6-6.7 ng/ ⁇ l (for example, 6.1-6.6 ng/ ⁇ l or 6.2-6.5 ng/ ⁇ l or 6.33 ⁇ 0.10 ng/ ⁇ l) of ATP as measured using the ATP Assay Kit (Sigma-Aldrich, MAK190).
  • Bacterial extracellular ATP can have pleiotropic effects including activation of T cell-receptor mediated signalling (Schenk et al., 2011), promotion of intestinal Th17 cell differentiation (Atarashi et al., 2008) and induction of secretion of the pro-inflammatory mediator IL-1(3 by activating the NLRP3 inflammasome (Karmarkar et al., 2016). Accordingly, a bacterial strain which is an extracellular ATP producer is useful for stimulating the immune system in the context of the method of the invention.
  • the bacterial strain for use in the invention comprises one or more of the following three genes: Mobile element protein; Xylose ABC transporter, permease component; and FIG00632333: hypothetical protein.
  • the bacterial strain for use in the invention comprises genes encoding Mobile element protein and Xylose ABC transporter, permease component; Mobile element protein and FIG00632333: hypothetical protein; Xylose ABC transporter, permease component and FIG00632333: hypothetical protein; or Mobile element protein, Xylose ABC transporter, permease component, and FIG00632333: hypothetical protein.
  • a particularly preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488.
  • This is the exemplary MRx0518 strain tested in the examples and shown to be effective for treating disease.
  • the invention provides, according to some embodiments, a bacterial composition as part of the invention, comprising a cell of the Enterococcus gallinarum strain deposited under accession number NCIMB 42488, or a derivative thereof.
  • a derivative of the strain deposited under accession number NCIMB 42488 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original.
  • a derivative of a strain of the composition comprised in the invention may be modified, for example at the genetic level, without ablating the biological activity.
  • a derivative strain of the invention is therapeutically active.
  • a derivative strain will have comparable immune modulatory activity to the original NCIMB 42488 strain.
  • a derivative strain will elicit comparable effects on the cancer disease models when which may be identified by using the culturing and administration protocols described in the Examples.
  • a derivative of the NCIMB 42488 strain will generally be a biotype of the NCIMB 42488 strain.
  • references to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 encompass any cells that have the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42488, and such cells are encompassed by the invention.
  • reference to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 refers only to the MRx0518 strain deposited under NCIMB 42488 and does not refer to a bacterial strain that was not deposited under NCIMB 42488.
  • reference to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 refers to cells that have the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42488, but which are not the strain deposited under NCIMB 42488.
  • Bacterial strains that are biotypes of the bacterium deposited under accession number 42761 are also expected to be effective for stimulating the immune system.
  • a biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.
  • Strains that are biotypes of the bacterium deposited under accession number NCIMB 42761 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 42761.
  • substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome).
  • a biotype strain has at least 98% sequence identity across at least 98% of its genome or at least 99% sequence identity across 99% of its genome.
  • Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42761. In some embodiments, a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0554 deposited as NCIMB 42761.
  • a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0554 deposited as NCIMB 42761 and has a 16S rRNA gene sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ ID NO:3.
  • a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0554 deposited as NCIMB 42761 and has the 16S rRNA gene sequence of SEQ ID NO:3.
  • strains that are biotypes of the bacterium deposited under accession number NCIMB 42761 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42761 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing.
  • FAFLP fluorescent amplified fragment length polymorphism
  • rep repetitive DNA element
  • strains that are biotypes of the bacterium deposited under accession number NCIMB 42761 and that are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 42761 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example, [15]).
  • ARDRA amplified ribosomal DNA restriction analysis
  • biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42761.
  • the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMérieux).
  • the bacterial strain used in the invention is:
  • the bacterial strain used in the invention is:
  • Enterococcus gallinarum strains that are useful in the compositions and methods of the invention, such as biotypes of the bacterium deposited under accession number NCIMB 42761, may be identified using any appropriate method or strategy, including the assays described in the examples.
  • strain for use in the invention may be identified by culturing in anaerobic YCFA and/or administering the bacteria to the type II collagen-induced arthritis mouse model and then assessing cytokine levels.
  • bacterial strains that have similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 42761 may be useful in the invention.
  • a useful strain will have comparable immune modulatory activity to the NCIMB 42761 strain.
  • a biotype strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
  • a derivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity.
  • a derivative strain of the invention is therapeutically active.
  • a derivative strain will have comparable immune modulatory activity to the original NCIMB 42761 strain.
  • a derivative strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
  • a derivative of the NCIMB 42761 strain will generally be a biotype of the NCIMB 42761 strain.
  • references to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42761 encompass any cells that have the same safety and therapeutic efficacy characteristics as the strain deposited under accession number NCIMB 42761, and such cells are encompassed by the invention.
  • reference to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42761 refers only to the MRx0554 strain deposited under NCIMB 42761 and does not refer to a bacterial strain that was not deposited under NCIMB 42761.
  • the bacterial strain for use in the invention has a genome with sequence identity to SEQ ID NO:2 OF WO2018/215782. In some embodiments, the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:2 OF WO2018/215782 across at least 60% (e.g. across at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:2 OF WO2018/215782.
  • sequence identity e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity
  • the bacterial strain for use in the invention may have a genome with at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 70% of SEQ ID NO:2 OF WO2018/215782, or at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 80% of SEQ ID NO:2 OF WO2018/215782, or at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 90% of SEQ ID NO:2 OF WO2018/215782, or at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 100% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 70% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 80% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2
  • the bacterial strain for use in the invention has a genome with sequence identity to SEQ ID NO:2 OF WO2018/215782, for example as described above, and a 16S rRNA gene sequence with sequence identity to SEQ ID NO:1 or 3, for example as described above, preferably with a 16S rRNA gene sequence that is at least 99% identical to SEQ ID NO:3, more preferably which comprises the 16S rRNA gene sequence of SEQ ID NO:3.
  • the bacterial strain for use in the invention has a genome with sequence identity to SEQ ID NO:2 OF WO2018/215782, for example as described above, and is effective for stimulating the immune system.
  • the bacterial strain for use in the invention has a genome with sequence identity to SEQ ID NO:2 OF WO2018/215782, for example as described above, and a 16S rRNA gene sequence with sequence identity to SEQ ID NO: 1 or 3, for example as described above, and is effective for stimulating the immune system.
  • the bacterial strain for use in the invention has a 16S rRNA gene sequence that is at least 99%, 99.5% or 99.9% identical to the 16S rRNA gene sequence represented by SEQ ID NO: 3 (for example, which comprises the 16S gene rRNA sequence of SEQ ID NO:3) and a genome with at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across at least 90% of SEQ ID NO:2 OF WO2018/215782, and which is effective for stimulating the immune system.
  • SEQ ID NO: 3 for example, which comprises the 16S gene rRNA sequence of SEQ ID NO:3
  • SEQ ID NO:3 for example, which comprises the 16S gene rRNA sequence of SEQ ID NO:3
  • the bacterial strain for use in the invention is a Enterococcus gallinarum and has a 16S rRNA gene sequence that is at least 99%, 99.5% or 99.9% identical to the 16S rRNA gene sequence represented by SEQ ID NO:3 (for example, which comprises the 16S rRNA gene sequence of SEQ ID NO:3) and a genome with at least 98% sequence identity (e.g. at least 99% or at least 99.5% sequence identity) to SEQ ID NO:2 OF WO2018/215782 across at least 98% (e.g. across at least 99% or at least 99.5%) of SEQ ID NO:2 OF WO2018/215782, and which is effective for stimulating the immune system.
  • SEQ ID NO:3 for example, which comprises the 16S rRNA gene sequence of SEQ ID NO:3
  • SEQ ID NO:3 for example, which comprises the 16S rRNA gene sequence of SEQ ID NO:3
  • a genome with at least 98% sequence identity e.g. at least 99% or at least
  • the bacterial strains in the compositions of the invention are viable and capable of partially or totally colonising the intestine.
  • the bacterial strain in the composition of the invention is of the species Enterococcus caselliflavus.
  • Enterococcus caselliflavus is highly similar to Enterococcus gallinarum and is also flagellated.
  • compositions of the invention can lead to immune stimulation. Since administration of the compositions of the invention were shown to have an immunostimulatory effect, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and diseases treatable by an increased immune response.
  • the compositions of the invention are for use in stimulating the immune system.
  • the compositions of the invention are for use in treating disease by stimulating the immune system.
  • the compositions of the invention are for use in promoting an immune response.
  • compositions of the invention may be useful in the treatment of diseases characterised by an increase in the percentage of Tregs in a cell population.
  • the compositions of the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of Tregs in a cell population.
  • the compositions of the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of CD4+CD25+CD127 ⁇ cells in a cell population.
  • the compositions of the invention are for use in treating or preventing diseases by decreasing the percentage of Tregs in cell populations.
  • compositions of the invention are for use in reducing suppression of the immune response by Tregs.
  • compositions of the invention are for use in stimulating the immune response by the selective reduction of Tregs.
  • compositions of the invention are for use in immunostimulation, wherein the compositions of the invention reduce the number or percentage of Tregs.
  • compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the ratio of CD8/Treg and/or activated CD8/Treg cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the ratio of CD8/Treg cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the ratio of activated CD8/Treg cells.
  • compositions of the invention are for use in stimulating the immune response by increasing the ratio of CD8/Treg cells.
  • compositions of the invention are for use in stimulating the immune response by increasing the ratio of activated CD8/Treg cells.
  • compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of B cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of B cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD19 + CD3 ⁇ cells.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of B cells in cell populations, wherein the increase in number or percentage of B cells results in immune stimulation.
  • compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of B cells.
  • compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8 T-cytotoxic cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD8 T-cytotoxic cells.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of CD8 T-cytotoxic cells in cell populations, wherein the increase in number or percentage of CD8 T-cytotoxic cells results in immune stimulation.
  • compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CD8 T-cytotoxic cells.
  • compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8 + activated cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD8 + activated cells.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of CD8 + activated cells in cell populations, wherein the increase in number or percentage of CD8 + activated cells results in immune stimulation.
  • compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CD8 + activated cells.
  • compositions of the invention can lead to an increase in expression of pro-inflammatory molecules, such as pro-inflammatory cytokines.
  • pro-inflammatory molecules such as pro-inflammatory cytokines.
  • pro-inflammatory molecules that showed an increase in expression levels upon administration of compositions of the invention include IL-8, IL-12p70, IL-23, TNF- ⁇ , IL-1 ⁇ , and IL-6. Since administration of the compositions of the invention were shown to increase the expression of pro-inflammatory molecules, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of pro-inflammatory molecules, such as pro-inflammatory cytokines.
  • compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of pro-inflammatory molecules, in particular diseases characterised by a decrease in the expression and/or activity of pro-inflammatory cytokines.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-8, IL-12p70, IL-23, TNF- ⁇ , IL-1 ⁇ ,- and/or IL-6.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-23, TNF- ⁇ , IL-1 ⁇ , and/or IL-6.
  • compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-8, IL-12p70, IL-23, TNF- ⁇ , IL-1 ⁇ , and/or IL-6.
  • compositions of the invention can lead to an increase in expression of IL-1 ⁇ .
  • IL-1 ⁇ is a pro-inflammatory cytokine [16].
  • the production and secretion of IL-1 ⁇ is regulated by the inflammasome, a protein complex which is associated with activation of the inflammatory response [17].
  • administration of the compositions of the invention were shown to increase the expression of IL-1 ⁇ , compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-1 ⁇ .
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-1 ⁇ .
  • the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-1 ⁇ .
  • compositions of the invention can lead to an increase in expression of IL-23.
  • IL-23 has been linked to inflammation [18,19].
  • the proposed functions of IL-23 in the immune response include promoting the proliferation of CD4 + memory T cells and promoting the secretion of IFN- ⁇ by dendritic cells (DCs) [20]. Since administration of the compositions of the invention were shown to increase the expression of IL-23, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-23. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-23.
  • compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-23. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-23.
  • TNF- ⁇ Tumour Necrosis Factor alpha
  • TNF- ⁇ is a pro-inflammatory cytokine which is known to be involved in various signalling pathways to promote cell death. TNF- ⁇ initiates apoptosis by binding to its cognate receptor, TNFR-1, which leads to a cascade of cleavage events in the apoptotic pathway [21]. TNF- ⁇ can also trigger necrosis via a RIP kinase-dependent mechanism [22].
  • compositions of the invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by TNF- ⁇ .
  • the compositions of the invention are for use in treating diseases characterised by decreased TNF- ⁇ expression.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of TNF- ⁇ .
  • the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of TNF- ⁇ .
  • compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of TNF- ⁇ .
  • compositions of the invention can lead to an increase in expression of IL-6.
  • IL-6 a pro-inflammatory cytokine that is produced during inflammation, and promotes the differentiation of na ⁇ ve CD4 + T cells and the differentiation of CD8 + T cells into cytotoxic T cells [23]. Since administration of the compositions of the invention were shown to increase the expression of IL-6, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-6.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-6.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-6.
  • compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-6.
  • compositions of the invention may selectively decrease the number or percentage of Tregs by increasing the expression of IL-6.
  • compositions of the invention are for use in immunostimulation by increasing the expression of IL-6.
  • compositions of the invention are for use in immunostimulation by decreasing the number or percentage of Tregs.
  • stimulating the immune system according to the present invention comprises TLR5 activation or upregulation of TLR5 activation. In some embodiments, stimulating the immune system according to the present invention comprises TLR9 activation or upregulation of TLR9 activation. In some embodiments, stimulating the immune system according to the present invention comprises activation of TLR5 and TLR9 or upregulation of TLR9 and TLR5 activation. In some embodiments, stimulating the immune system according to the present invention comprises inducing and/or upregulating differentiation of T cells such as, but not limited to, T helper cells and T cytotoxic cells.
  • TLR signalling pathways culminate in the activation of the transcription factor nuclear factor-kappaB (NF- ⁇ B).
  • NF- ⁇ B controls the expression of an array of inflammatory cytokine genes, including TNF- ⁇ .
  • Immune stimulation causes, for example, the dimerization of TLR5, which subsequently recruits MyD88 and activates protein kinases, including IRAK1, IRAK2, IRAK4 and IRAN-M.
  • the activation of these kinases leads to the nuclear localization of NF- ⁇ B, which is a proinflammatory cytokine [25].
  • compositions of the invention lead to an increase in expression of NF- ⁇ B. Since administration of the compositions of the invention increase the expression of the proinflammatory cytokine NF- ⁇ B, compositions of the invention may be useful in stimulating the immune response. In addition, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and/or diseases treatable by an increased immune response. In one embodiment, the compositions of the invention are for use as an immune stimulant by increasing the level and/or activity of NF- ⁇ B. In one embodiment, the compositions of the invention are for use in treating diseases characterised by reduced immune activation by increasing the level and/or activity of NF- ⁇ B. In one embodiment, the compositions of the invention are for use in treating diseases treatable by an increased immune response by increasing the level and/or activity of NF- ⁇ B.
  • compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression and/or activation of NF- ⁇ B.
  • the compositions of the invention are for use in treating diseases characterised by a decrease in expression and/or activation of NF- ⁇ B.
  • compositions of the invention are likely to be useful as adjuvants to treat infectious diseases, allergies and tumours by promoting both innate and adaptive immune responses [25].
  • the compositions of the invention are for use in treating infectious diseases, allergies and/or tumours.
  • the compositions of the invention are for use in treating infectious diseases, allergies and/or tumours by increasing the level and/or activity of NF- ⁇ B.
  • compositions of the invention promote the differentiation of T-helper cells and cytotoxic T lymphocytes. Therefore, in certain embodiments, the compositions of the invention are for use in stimulating the differentiation of T-helper cells and/or cytotoxic T lymphocytes. In certain embodiments, the disease to be treated by the compositions of the invention is not cancer.
  • compositions of the invention can lead to an increase in expression of Tumour Necrosis Factor alpha (TNF- ⁇ ).
  • TNF- ⁇ Tumour Necrosis Factor alpha
  • TNF- ⁇ is known to be important for vaccine responses.
  • TNF- ⁇ has been shown to be required for an efficient vaccine response in a flu vaccination of the elderly population [26]. Since administration of the compositions of the invention were shown to increase TNF- ⁇ expression, compositions of the invention may be useful as a vaccine adjuvant.
  • the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of TNF- ⁇ .
  • the compositions of the invention are for use as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant in influenza therapy. In certain embodiments, the compositions of the invention are for use in enhancing an immune response against an antigen. In certain embodiments, the invention provides a composition to be administered in combination with an antigen. In certain embodiments, the compositions of the invention are for administration to a patient shortly prior to or after vaccination.
  • Enterococcus gallinarum and in particular strain MRx0518 is flagellated and flagellins can be TLR5 agonists.
  • TLR agonists are in development as vaccine adjuvants across a range of antigen types, particularly in the elderly population [27].
  • the data in the examples confirm that MRx0518 flagellin is a TLR5 agonists. Therefore, the compositions of the invention may be useful as vaccine adjuvants, in particular for vaccine administered to elderly patients (e.g. over 40, 50, 60, 70 or 80 years of age), who may have reduced immune system activity.
  • TLR5 signalling also plays a key role in age-associated innate immune responses [28].
  • the compositions are for use in enhancing an innate immune response.
  • TLR5 agonists are in development as vaccine adjuvants, these are all from known pathogens and/or synthetic.
  • the compositions of the invention comprise commensal bacteria.
  • compositions of the invention can lead to an increase in expression of IL-6.
  • Increased IL-6 expression has been associated with vaccine responses for many diseases.
  • IL-6 was produced by CD14+CD16 ⁇ inflammatory monocytes after adults were administered an influenza vaccine [29], and higher levels of IL-6 were associated with achieving a vaccine response to an influenza vaccine [30].
  • IL-6 was produced after injection of the AS03 adjuvant system [31] and downregulation of IL-6 in mice was shown to reduce the helper T cell response after administration of a tuberculosis vaccine [32]. Since administration of the compositions of the invention were shown to increase IL-6 expression, compositions of the invention may be useful as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in tuberculosis therapy.
  • compositions of the invention may be useful as a vaccine adjuvant.
  • compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF- ⁇ . In one embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of IL-6. In a particular embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF- ⁇ and IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in HIV therapy. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in chlamydia therapy.
  • compositions of the invention can lead to an increase in expression of IL-1 ⁇ .
  • Li et al. [35] showed that the adjuvant aluminium hydroxide activated the secretion of IL-1 ⁇ , and suggested that IL-1 ⁇ itself can act as an adjuvant. Since administration of the compositions of the invention were shown to increase IL-1 ⁇ expression, compositions of the invention may be useful as a vaccine adjuvant.
  • administration of the compositions of the invention can increase the ratio of CD8 + T cells to Tregs.
  • compositions of the invention may be useful as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant.
  • the compositions of the invention are for use as a vaccine adjuvant by increasing the ratio of CD8 + T cells to Tregs.
  • compositions of the invention can lead to an increase in expression or levels of CXCR3 ligands CXCL9 and CXCL10.
  • Known adjuvants such as ASO3, CpG, GLA-SE, ⁇ GalCer all increase CXCL9 and 10 [37,38], which suggests the compositions of the invention will be effective as adjuvants.
  • CXCL9 and 10 are associated with IFN ⁇ /Th1 responses and promote antibody responses [39].
  • the compositions of the invention are for use in promoting an antibody response against an antigen, in particular a pathogenic or cancer antigen.
  • CXCL9 is a more sensitive measure than IFN- ⁇ of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines [40].
  • compositions of the invention are for use in promoting an T-cell response against an antigen, in particular a pathogenic or cancer antigen.
  • compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of CXCL9 and CXCL10.
  • the compositions are for use in protecting against malaria.
  • compositions of the invention can lead to an increase in expression or levels of IL-12p70.
  • This effect has been associated with vaccine adjuvant efficiency and IL-12 has been proposed as an adjuvant itself [41], which suggests the compositions of the invention will be effective as adjuvants.
  • the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-12p70.
  • compositions of the invention when used as a vaccine adjuvant, will be administered on their own to provide an adjuvant effect for an antigen that has been separately administered to the patient.
  • the composition of the invention is administered orally, whilst the antigen is injected parenterally.
  • compositions of the invention may be used for enhancing an immune response to any useful antigen.
  • antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumour antigens.
  • the invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomegalovirus, Staphylococcus aureus, chlamydia , SARS coronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis , Epstein Barr virus, human papillomavirus, etc.
  • antigens for use with the invention include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol or opiates.
  • MAGE Carcinoembryonic antigen
  • CEA Carcinoembryonic antigen
  • MUC-1 MUC-1
  • HER2 sialyl-Tn
  • STn sialyl-Tn
  • hTERT human telomerase reverse transcriptase
  • WT1 Wilms tumour gene
  • CA-125 CA-125
  • PSA
  • Preferred antigens for use with the invention include pathogen antigens and tumour antigens.
  • An antigen will elicit an immune response specific for the antigen that will be effective for protecting against infection with the pathogen or attacking the tumour.
  • Antigens may be, for example, peptides or polysaccharides.
  • the invention also provides the use of: (i) an aqueous preparation of an antigen; and (ii) a composition comprising a bacterial strain of the species Enterococcus gallinarum , in the manufacture of a medicament for raising an immune response in a patient.
  • the immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response.
  • a bacterial strain of the species Enterococcus gallinarum is engineered to present an antigen. Presenting an antigen on the bacterial strain of the invention may maximise the immunostimulatory activities and further enhance the protective immune response generated against the antigen.
  • manufacturing and delivering therapeutics comprising an antigen and a bacteria of the invention may be more efficient and effective this way than when each of the antigen and the composition comprising the bacterial strain are manufactured and administered separately. Therefore, in some embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum that presents an antigen, for example on its cell surface.
  • the composition comprising the bacterial strain that presents an antigen is for use as a vaccine antigen.
  • the antigen is derived from HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomegalovirus, Staphylococcus aureus, chlamydia , SARS coronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis , Epstein Barr virus or human papillomavirus.
  • the antigen is a glycoprotein antigen, lipoglycan antigen, archaea antigen, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 or a habit forming substance, such as, alcohol, opiates and the like.
  • MAGE Carcinoembryonic antigen
  • CEA Carcinoembryonic antigen
  • MUC-1 MUC-1
  • HER2 sialyl-Tn
  • STn sialyl-Tn
  • hTERT human telomerase reverse transcriptase
  • WT1 Wilms tumour gene
  • CA-125 CA-125
  • the bacteria of the invention express one or more antigens.
  • the antigen will be expressed recombinantly and will be heterologous to the bacteria of the invention. Therefore, the invention provides a bacterial strain of the species Enterococcus gallinarum that expresses a heterologous antigen.
  • the antigen may be part of a fusion polypeptide expressed with one or more polypeptides homologous to the bacteria.
  • the bacteria express the antigen as a non-fusion polypeptide.
  • the invention provides a composition comprising a cell of a bacterial strain of the species Enterococcus gallinarum , wherein the cell expresses a heterologous antigen.
  • the composition is for use as a vaccine.
  • the invention provides a cell of a bacterial strain of the species Enterococcus gallinarum , wherein the cell expresses a heterologous antigen.
  • the cell is for use as a vaccine.
  • antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens.
  • Further antigens for expressing in a bacterial strain of the species Enterococcus gallinarum include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol, opiates, or the like.
  • MAGE Carcinoembryonic antigen
  • the invention may also be useful for enhancing the response to vaccines against non-communicable diseases such as elevated cholesterol (e.g. via the PCSK9 antigen).
  • the invention may also be useful for enhancing the response to vaccines against habit forming substances, for example nicotine, alcohol or opiates.
  • compositions of the invention can lead to an increase in expression of IL-6.
  • Increased IL-6 expression has been correlated with response to CD19 CAR-T therapy of chronic lymphocyte leukaemia.
  • An increase in serum IL-6 was associated with CAR-T cell expansion, whereas inhibition of IL-6 was associated with inhibition of CAR-T cell proliferation [42].
  • administration of the compositions of the invention were shown to increase IL-6 expression, compositions of the invention may be useful in cell therapy, in particular CAR-T cell therapy.
  • the compositions of the invention are for use in cell therapy.
  • the compositions of the invention are for use in CAR-T cell therapy.
  • compositions of the invention are for use in the treatment of chronic lymphocyte leukaemia.
  • compositions of the invention may be useful in cell therapy, in particular CAR-T cell therapy.
  • compositions of the invention may be useful in cell therapy, in particular in enhancing the response to a cell therapy.
  • MSC Mesenchymal stem cell
  • compositions of the invention may be useful for stem cell differentiation in stem cell transplantation therapy.
  • Hematopoietic stem cell transplantation is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. Colonisation of the gut with Enterococci ( Enterococcus gallinarum and Enterococcus casseliflavus ) prior to allogenic hematopoietic stem cell transplantation has been shown to lead to a significantly improved the 2-year survival of patients after due to decreased nonrelapse mortality [46]. Therefore, the immunomodulatory effect shown in the examples may be useful in hematopoietic stem cell transplantation therapy. In certain embodiments, the compositions of the invention may be useful in improving survival after hematopoietic stem cell transplantation and in particular after allogenic hematopoietic stem cell transplantation.
  • compositions of the invention may be useful in combination with allogenic hematopoietic stem cell transplantation.
  • the compositions of the invention may be effective in boosting successful patient response to allogenic hematopoietic stem cell transplantation.
  • the compositions of the invention are administered prior to hematopoietic stem cell transplantation.
  • the compositions of the invention are for administration to a patient scheduled to receive hematopoietic stem cell transplantation.
  • the compositions of the invention are administered following hematopoietic stem cell transplantation.
  • the compositions of the invention are for administration to a patient that has received hematopoietic stem cell transplantation.
  • compositions of the invention may be used to prevent or delay immunosenescence.
  • compositions of the invention are for use in preventing immunosenescence.
  • compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number.
  • compositions of the invention are for use in delaying immunosenescence characterised by a decrease in B cell number.
  • compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number and a decrease in B cell number.
  • compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number. In one embodiment, compositions of the invention are for use in delaying immunosenescence by increasing B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number and increasing B cell number. In one embodiment, compositions of the invention are for use in treating diseases caused by immunosenescence. In one embodiment, compositions of the invention are for use in treating aging-related diseases by delaying and/or preventing immunosenescence.
  • compositions of the invention may be useful for preventing or delaying immunosenescence.
  • compositions of the invention are for use in delaying and/or preventing immunosenescence as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant, wherein the compositions delay and/or prevent immunosenescence.
  • compositions of the invention are to be administered to the gastrointestinal tract in order to enable delivery to and/or partial or total colonisation of the intestine with the bacterial strain of the invention.
  • compositions of the invention are administered orally (including sublingual), but they may be administered rectally or intranasally.
  • compositions of the invention may be administered as a foam, as a spray or a gel.
  • compositions of the invention may be administered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
  • a rectal suppository for example in the form of a theobroma oil (coa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
  • the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
  • a tube such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
  • compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily.
  • treatment according to the invention is accompanied by assessment of the patient's gut microbiota. Treatment may be repeated if delivery of and/or partial or total colonisation with the strain of the invention is not achieved such that efficacy is not observed, or treatment may be ceased if delivery and/or partial or total colonisation is successful and efficacy is observed.
  • composition of the invention may be administered to a pregnant animal, for example a mammal such as a human in order to reduce the likelihood of disease developing in her child in utero and/or after it is born.
  • compositions of the invention may be administered to a patient that has been diagnosed with a disease or condition mediated reduced immune activity, or that has been identified as being at risk of a disease or condition mediated by reduced immune activity.
  • the compositions may also be administered as a prophylactic measure to prevent the development of diseases or conditions mediated by reduced immune activity in a healthy patient.
  • compositions of the invention may be administered to a patient that has been diagnosed with deficient immune activity, or that has been identified as being at risk of deficient immune activity.
  • the patient may have reduced or absent colonisation by Enterococcus , and in particular Enterococcus gallinarum.
  • compositions of the invention may be administered as a food product, such as a nutritional supplement.
  • compositions of the invention are for the treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits.
  • the compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used.
  • the composition of the invention comprises bacteria.
  • the composition is formulated in freeze-dried form.
  • the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain of the invention.
  • the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [51,52].
  • composition of the invention may comprise a live, active bacterial culture.
  • the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine.
  • Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [53] and [54].
  • the composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Enterococcus are anaerobes. Other ingredients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and/or partial or total colonisation and survival in vivo.
  • the probiotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.
  • composition may be formulated as a probiotic.
  • a composition of the invention includes a therapeutically effective amount of a bacterial strain of the invention.
  • a therapeutically effective amount of a bacterial strain is sufficient to exert a beneficial effect upon a patient.
  • a therapeutically effective amount of a bacterial strain may be sufficient to result in delivery to and/or partial or total colonisation of the patient's intestine.
  • a suitable daily dose of the bacteria may be from about 1 ⁇ 10 3 to about 1 ⁇ 10 11 colony forming units (CFU); for example, from about 1 ⁇ 10 7 to about 1 ⁇ 10 10 CFU; in another example from about 1 ⁇ 10 6 to about 1 ⁇ 10 10 CFU; in another example from about 1 ⁇ 10 7 to about 1 ⁇ 10 11 CFU; in another example from about 1 ⁇ 10 8 to about 1 ⁇ 10 10 CFU; in another example from about 1 ⁇ 10 8 to about 1 ⁇ 10 11 CFU.
  • CFU colony forming units
  • the dose of the bacteria is at least 10 9 cells per day, such as at least 10 10 , at least 10 11 , or at least 10 12 cells per day.
  • the composition contains the bacterial strain in an amount of from about 1 ⁇ 10 6 to about 1 ⁇ 10 11 CFU/g, respect to the weight of the composition; for example, from about 1 ⁇ 10 8 to about 1 ⁇ 10 10 CFU/g.
  • the dose may be, for example, 1 g, 3 g, 5 g, and 10 g.
  • the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 ⁇ 10 3 to about 1 ⁇ 10 11 colony forming units per gram with respect to a weight of the composition.
  • the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg or between 750 mg and 1000 mg.
  • the invention provides the above pharmaceutical composition, wherein the lyophilised bacteria in the pharmaceutical composition is administered at a dose of between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg or between 750 mg and 1000 mg.
  • a probiotic such as the composition of the invention, is optionally combined with at least one suitable prebiotic compound.
  • a prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract.
  • Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides.
  • the probiotic composition of the present invention includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight).
  • Carbohydrates may be selected from the group consisting of: fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans, arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers.
  • the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.
  • compositions of the invention may comprise pharmaceutically acceptable excipients or carriers.
  • suitable excipients may be found in the reference [55].
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [56].
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • compositions of the invention may be formulated as a food product.
  • a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement.
  • a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition.
  • the composition of the invention is formulated as a milk-based product.
  • milk-based product means any liquid or semi-solid milk- or whey-based product having a varying fat content.
  • the milk-based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products.
  • milk beverages such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.
  • compositions of the invention contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism.
  • compositions for use in accordance with the invention may or may not require marketing approval.
  • the lyophilised bacterial strain is reconstituted prior to administration.
  • the reconstitution is by use of a diluent described herein.
  • compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder when administered to a subject in need thereof.
  • the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition.
  • the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition.
  • the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition.
  • the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition mediated by pro-inflammatory cytokines, such as IL-1 ⁇ , TNF- ⁇ , MIP-3 ⁇ , IL-23 or IL-6.
  • the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition mediated by TNF- ⁇ .
  • the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 ⁇ 10 3 to about 1 ⁇ 10 11 colony forming units per gram with respect to a weight of the composition.
  • the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.
  • the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.
  • the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.
  • the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.
  • the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
  • an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
  • the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.
  • the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.
  • the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4.0 or about 25.0 and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.
  • the bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [57,58].
  • the solid or liquid medium used for culture may be YCFA agar or YCFA medium.
  • YCFA medium may include (per 100 mL, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO 3 (0.4 g), cysteine (0.1 g), K 2 HPO 4 (0.045 g), KH 2 PO 4 (0.045 g), NaCl (0.09 g), (NH 4 ) 2 SO 4 (0.09 g), MgSO 4 .7H 2 O (0.009 g), CaCl 2 ) (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 ⁇ g), cobalamin (1 ⁇ g), p-aminobenzoic acid (3 ⁇ g), folic acid (5 ⁇ g), and pyridoxamine (15 ⁇ g).
  • the compositions of the invention may also be useful for preventing diseases or conditions, when administered as vaccine compositions.
  • the bacterial strains of the invention may be killed, inactivated or attenuated.
  • the compositions may comprise a vaccine adjuvant.
  • the compositions are for administration via injection, such as via subcutaneous injection.
  • composition “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
  • references to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [62].
  • a preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith-Waterman homology search algorithm is disclosed in ref. [63].
  • a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.
  • compositions comprising bacterial strains according to the invention tested the efficacy of compositions comprising bacterial strains according to the invention in four tumor models.
  • Test substance Bacterial strain #MRx0518.
  • Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131, isotype: Syrian Hamster IgG1, Bioxcell).
  • Test and reference substances vehicles Bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each day of injection to mice, antibody was diluted with PBS (ref: BE14-516F, Lonza, France).
  • Treatment doses Bacteria: 2 ⁇ 10 8 in 200 pt.
  • the anti-CTLA-4 was injected at 10 mg/kg/inj.
  • Anti-CTLA-4 was administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse weighing 20 g, 200 ⁇ L of test substance will be administered) according to the most recent body weight of mice.
  • Culture conditions of bacterial strain were as follows:
  • the EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule [64].
  • the LL/2 (LLC1) cell line was established from the lung of a C57BL/6 mouse bearing a tumour resulting from an implantation of primary Lewis lung carcinoma [65].
  • the Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse [66].
  • adherent tumour cells were detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.
  • BALB/C mice Healthy female BALB/C mice, of matching weight and age, were obtained from CHARLES RIVER for the EMT6 and RENCA model experiments.
  • C57BL/6 mice Healthy female C57BL/6 (C57BL16J) mice, of matching weight and age, were obtained from CHARLES RIVER (L'Arbresles) for the LL/2(LLC1) and the Hepa1-6 model experiments.
  • Animals were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals were followed [67,68]. Animals were maintained in housing rooms under controlled environmental conditions: Temperature: 22 ⁇ 2° C., Humidity 55 ⁇ 10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation.
  • Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cm 2 cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, water from water bottles.
  • group housing 900 cm 2 cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, water from water bottles.
  • mice from the positive control group received anti-CTLA-4 antibody treatments.
  • tumours were induced by subcutaneous injection of 1 ⁇ 10 6 EMT-6 cells in 200 ⁇ L RPMI 1640 into the right flank of mice.
  • Euthanasia Each mouse was euthanized when it reached a humane endpoint as described below, or after a maximum of 6 weeks post start of dosing.
  • mice from the positive control group received anti-CTLA-4 antibody treatments.
  • Euthanasia Each mouse was euthanized when it reached a humane endpoint as described below, or after a maximum of 6 weeks post start of dosing.
  • mice from the positive control group received anti-CTLA-4 antibody treatments.
  • Hepa 1-6 tumor cells in animals by intrasplenic injection On D0, one million (1 ⁇ 10 6 ) Hepa 1-6 tumor cells in 50 ⁇ L RPMI 1640 medium were transplanted via intra-splenic injection into mice. Briefly, a small left subcostal flank incision was made and the spleen was exteriorized. The spleen was exposed on a sterile gauze pad, and injected under visual control with the cell suspension with a 27-gauge needle. After the cell inoculation, the spleen was excised.
  • Euthanasia Each mouse was euthanized when it reached a humane endpoint as described in section below, or after a maximum of 6 weeks post start of dosing.
  • Treatment schedule The start of first dosing was considered as D0. On D0, non-engrafted mice were randomized according to their individual body weight into groups of 12 mice. On D0, the mice received vehicle (culture medium) or bacterial strain (2 ⁇ 10 8 in 100 ⁇ L, PO). On D14, all mice were engrafted with RENCA tumour cells injected SC into the left hind flank as described below. Treatment with anti-CTLA-4 (clone 9D9, 10 mg/kg, IP) and anti-PDL1 (clone 10F.9G2, 10 mg/kg, IP) was initiated from D17.
  • CTLA-4 clone 9D9, 10 mg/kg, IP
  • anti-PDL1 clone 10F.9G2, 10 mg/kg, IP
  • Euthanasia Each mouse was euthanized when it reached a humane endpoint as described in section below, or after a maximum of 6 weeks post start of dosing.
  • tumour burden at euthanasia At the time of termination, tumours were collected and their volume evaluated.
  • Tumor ⁇ ⁇ volume ⁇ width 2 ⁇ length 2
  • Humane endpoints [70]: Signs of pain, suffering or distress: pain posture, pain face mask, behaviour; Tumor exceeding 10% of normal body weight, but non-exceeding 2000 mm 3 ; Tumors interfering with ambulation or nutrition; Ulcerated tumour or tissue erosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, emaciation, cachexia, dehydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia, significant bleeding; Neurologic signs: circling, convulsion, paralysis; Sustained decrease in body temperature; Abdominal distension.
  • Anaesthesia Isoflurane gas anesthesia was used for all procedures: surgery or tumour inoculation, i.v. injections, blood collection. Ketamine and Xylazine anesthesia was used for stereotaxia surgical procedure.
  • Analgesia Carprofen or multimodal carprofen/buprenorphine analgesia protocol were adapted to the severity of surgical procedure. Non-pharmacological care was provided for all painful procedures. Additionally, pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending veterinarian.
  • Euthanasia Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.
  • FIG. 1A The results are shown in FIG. 1A .
  • Treatment with the bacterial strain of the invention led to a clear reduction in tumour volume relative to both the negative controls.
  • the positive control also led to a reduction in tumour volume, as would be expected.
  • MRx0518 conveys its therapeutic effects in syngeneic tumour models
  • ex vivo analysis was performed on the syngeneic EMT6 tumour model studies. While tumour volume is the primary measurement in preclinical oncology studies, tumours often consist of actively dividing tumour cells along with a necrotic core.
  • MRx0518 treatment had influence on the degree of necrosis found within EMT6 tumours, paraffin sections from the mid-belly region of the tumours were stained with Haematoxylin and Eosin.
  • MRx0518 treatment of a murine EMT6 breast carcinoma model showed a tendency towards increasing the cross-sectional area of necrosis within the tumour ( FIG. 1B , upper panel).
  • MRx0518 treatment had influence on dividing cells within the tumour.
  • paraffin sections from the mid-belly region of the tumours were stained with the proliferation protein Ki67, along with DAPI counter stain, to estimate the percentage of cells dividing within the EMT6 tumour.
  • tumour microenvironment was performed through flow cytometry of the tumour, to investigate the hypothesis that the MRx0518 bacterial strain has the ability to regulate the immune system into inducing an anti-tumour effect.
  • Tumours excised from the different treatment groups were cut into pieces. One piece was subjected to flow cytometry analysis. To assess the relative percentage of T lymphocytes, present within the tumours, the following markers were used: CD45, CD3, CD4, CD8, CD25 and FoxP3.
  • the preliminary flow cytometry data presented in FIG. 1C shows that the relative percentage of lymphocytes in tumours was slightly decreased in both the MRx0518 and anti-CTLA-4 treated groups, when compared respectively to vehicle or control animals. Likewise, the relative percentage of CD4 + cells appeared to be decreased in MRx0518 and anti-CTLA-4 treated animals, whilst the relative percentage of CD8 + cells followed an opposite trend in both groups, albeit with different magnitude.
  • the relative percentage of CD4 + FoxP3 + cells was lower in the anti-CTLA-4 treated group when compared to the slight decrease in MRx0518 treated animals; however, the reduction in the relative percentage of CD4 + CD25 + cells was noticeable only in the anti-CTLA-4 treated group.
  • the CD8+/FoxP3+ ratio showed a greater increase in the anti-CTLA-4 treated group than in the MRx0518 animals.
  • the NanoString data shows that the abundance of B cells, CD45, T cells, cytotoxic and NK cells were increased in the tumour tissue of MRx0518-treated group when compared to vehicle-treated animals ( FIG. 23 ).
  • the data presented here supports the hypothesis that MRx0518 has an immunostimulatory effect by increasing leukocytes, in particular NK cells, T cells and cytotoxic cells in the tumour microenvironment.
  • tumour piece was used for total protein extraction and subsequent cytokine analysis, together with plasma samples.
  • Protein levels of IL-10, CXCL1, CXCL2, CXCL10, IL-1 ⁇ , IL-17A, GM-CSF, TNF- ⁇ , IL-12p70 and IFN- ⁇ in the tumour microenvironment were analysed by MagPix technology. While IL-17A and GM-CSF were below levels of detection, all the other markers were expressed at reasonable levels ( FIG. 1D ). A significance difference was observed between the vehicle and anti-CTLA-4 group for IFN- ⁇ . The production of the IL-10 and IL-12p70 immune markers seemed reduced following MRx0518 treatment compared to the control treatments.
  • Cytokine levels were also assessed in blood plasma of the same animals. Protein levels of IL-23, IL-6, IL-10, VEGF, CXCL1, CXCL2, CXCL10, IL-2, IL-1 ⁇ , IL-17A, GM-CSF, TNF- ⁇ , IL-12p70 and IFN- ⁇ were analysed by MagPix technology. Overall, little cytokine production was detected in the blood plasma of animals either before tumour induction or at the end of the study ( FIG. 1E ). VEGF and CXCL10 were detected at substantial levels, while IL-23, IL-6, IL-10, CXCL1 and CXCL2 were detected at low levels.
  • IL-2, IL-lb, IL-17A, GM-CSF, TNF- ⁇ , IL-12p70 and IFN- ⁇ were not detected in the samples.
  • MRx0518 significantly increased production of IL-6 at Day 0.
  • MRx0518 also seemed to increase IL-23 production.
  • VEGF and CXCL10 were significantly downregulated in the anti-CLTA-4 group at Day 22.
  • the differences in cytokine production in the tumour and plasma, between MRx0518 and ant-CTLA-4 suggests that each of them acts on a distinct and potentially complementary mechanism.
  • cryostat CM 1950 Leica
  • the sections were then air-dried for 1 hour, fixed for 10 minutes in ice-cold methanol, washed in PBS, blocked in 10% BSA in PBS pH 7.2 before being incubated overnight with the primary antibody (rat-anti-mouse-CD8 ⁇ antibody, Sigma-Aldrich, Millipore).
  • mice 53 5 MRx0518 70 7 Anti-CTLA4 71 8
  • Ileum cryosections stained with anti-CD8 ⁇ showed a higher number of CD8 ⁇ positive cells localized in the crypt region tissues from animals treated with MRx0518 and anti-CTLA-4 compared to the vehicle group.
  • the results are shown in FIG. 3A .
  • the untreated negative control does not appear as would be expected, because liver weight was lower in this group than the other groups.
  • the vehicle negative control and the positive control groups both appear as would be expected, because mice treated with vehicle alone had larger livers than mice treated with anti-CTLA4 antibodies, reflecting a greater tumour burden in the vehicle negative control group.
  • Treatment with the bacterial strain of the invention led to a clear reduction in liver weight (and therefore tumour burden) relative to the mice in the vehicle negative control group.
  • strain MRx0518 may be useful for treating or preventing other diseases associated with reduced immune system activity.
  • MRx0518 A pure culture of bacteria MRx0518 was studied in a PCR gene analysis. There were two arms to the experiment: 1) MRx0518 was co-cultured with human colonic cells (CaCo2) to investigate the effects of the bacteria on the host, and 2) MRx0518 was co-cultured on CaCo2 cells that were stimulated with IL1 to mimic the effect of the bacteria in an inflammatory environment. The effects in both scenarios were evaluated through gene expression analysis. The results are shown below:
  • CXCL9 34.76 CXCR3 ligand primarily thought of as Th1 cell chemoattractant (inducible by IFN-g) IL8 31.81 Cytokine, chemoattractant (especially neutrophils), many receptors including CXCR1 and CXCR2/ CXCL1 16.48 CXCR2 ligand, stimulates cell proliferation as well as migration, overexpression is neuroprotective in EAE.
  • TNF 13.50 Major proinflammatory cytokine IL17C 12.18 Promotes antibacterial response from epthielium, synergistic with IL-22, CXCL10 10.66 Close homology with CXCL9, think also CXCR3 ligand? HSPA1B 10.19 Heat shock protein NFKBIA 8.87 NF ⁇ B signalling; PI3K JUN 7.61 Antibacterial response; GPCR signalling.
  • IL32 4.29 Proinflammatory cytokine, induced by IFN-g, IL-18 DUSP5 3.12 Anti-inflammatory, inactivates MAPK FOS 3.03 Transcription factors, TLR signalling, forms part of AP-1 GADD45B 2.89 Cell growth and proliferation CLDN4 2.61 Tight junctions ADM 2.57 NF ⁇ B signalling KLF10 2.49 Cell arrest, TGF-b signaling.
  • CXCR1/2 ligands CXCL3, CXCL2, CXCL1, IL-8
  • CXCR3 ligands CXCL9, CXCL10
  • a composition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25° C. or 4° C. and the container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols.
  • a monocyte population was isolated from peripheral blood mononuclear cells (PBMCs).
  • the monocyte cells were subsequently differentiated into immature dendritic cells.
  • the immature dendritic cells were plated out at 200,000 cells/well and incubated with MRx0518 at a final concentration of 10 7 /mL, with the optional addition of LPS at a final concentration of 100 ng/mL.
  • the negative control involved incubating the cells with RPMI media alone and positive controls incubated the cells with LPS at a final concentration of 100 ng/mL.
  • the cytokine content of the cells was then analysed.
  • FIGS. 4 a - d The results of these experiments can be seen in FIGS. 4 a - d .
  • the addition of MRx0518 alone leads to a substantial increase in the level of cytokines IL-6 and TNF- ⁇ compared to the negative control ( FIGS. 4 a and c ).
  • the addition of LPS (positive control) leads to an increase in the level of IL-6 and TNF- ⁇ compared to the negative control but not IL-1 ⁇ ( FIG. 4 b ).
  • a combination of MRx0518 and LPS led to a synergistic increase in the level of IL-1 ⁇ produced ( FIG. 4 d ).
  • MRx0518 has the ability to induce higher IL-6 and TNF- ⁇ cytokine production in immature dendritic cells.
  • the combination LPS and MRx0518 can increase the levels of cytokines IL-1 ⁇ in immature dendritic cells.
  • THF-1 cells were differentiated into MO medium for 48 h with 5 ng/mL phorbol-12-myristate-13-acetate (PMA). These cells were subsequently incubated with MRx0518 at a final concentration of 10 8 /mL, with or without the addition of LPS at a final concentration of 100 ng/mL. The bacteria were then washed off and the cells allowed to incubate under normal growing conditions for 24 h. The cells were then spun down and the resulting supernatant was analysed for cytokine content.
  • PMA phorbol-12-myristate-13-acetate
  • MRx0518 has the ability to induce cytokine production in THP-1 cells, which can be synergistically increased with the addition of LPS. These data indicate that MRx0518 alone or in combination with LPS can increase inflammatory cytokines IL-1 ⁇ , IL-6 and TNF- ⁇ , which promotes inflammation.
  • the inventors sought to further analyse the immunostimulatory effect of compositions of the invention.
  • the inventors analysed the expression of particular cytokines from THP-1 macrophages and dendritic cells derived from monocytes upon treatment with MRx0518. Macrophages and dendritic cells are key components of the innate immune system, act as messengers between the innate and adaptive immune systems and are resent in the gut where they release a variety of cytokines to modulate the immune response.
  • Cytokines involved in the innate immune response were analysed and also cytokines involved in the recruitment and activation of adaptive immune cells (IL-8, IL-23, IL-1 ⁇ and IL-6).
  • MRx0518 induces a strong and characteristic immuno-stimulatory profile in THP-1-derived macrophages and DCs derived from monocytes. Cytokines involved in the innate immune response (TNF- ⁇ , IL-12 and IL-10) are significantly induced by MRx0518 in both DCs and macrophages. MRx0518 induces a very strong and significant induction of IL-8 in both macrophages and DCs. MRX0581 induces a strong and significant induction of IL-23 and IL-6. MRx0518 also induced IL-1 ⁇ .
  • MRx0518 has immunostimulatory properties, and may be an effective composition for immunostimulation.
  • MRx0518 stimulates the immune system.
  • a TLR5 signalling reporter assay was selected and the data are presented in FIGS. 14 and 15 .
  • MRx0518 supernatant was the most potent activator of TLR5 and NF- ⁇ B. Also, the supernatant was treated with various lytic enzymes and trypsin was found to abrogate the majority of activity.
  • Example 8 Adjuvant Immunostimulatory Activity of MRx0518 in a Therapeutic Combination with a CTLA-4 Inhibitor
  • Test and reference substances Bacterial strain #MRx0518; Anti-CTLA4 antibody (ref: BE0131, Bioxcell; clone: 9H10; reactivity: mouse; isotype: Hamster IgG1; storage conditions: +4° C.).
  • Test and reference substances vehicles The MRx0518 bacteria were grown in a bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)) and kept as a glycerol stock at ⁇ 80° C. The animals were dosed with the bacteria according to the study protocol.
  • the anti-CTLA-4 antibodies were diluted with PBS (ref: BE14-516F, Lonza, France) on each day of injection to mice.
  • Treatment doses Bacteria: 2 ⁇ 10 8 in 200 ⁇ L.
  • the anti CTLA4 antibodies were administered at 10 mg/kg body weight according to the most recent body weight of mice.
  • the bacterial composition was administered by oral gavage (per os, PO) via a gavage tube at a volume of 200 ⁇ L/inj.
  • the anti CTLA-4 antibodies were injected into the peritoneal cavity of mice (Intraperitoneally, IP) at a volume of 10 mL/kg adjusted to the most recent individual body weight of mice.
  • the cell line that was used in this study is the EMT-6 cell line that was obtained from the ATCC (American Type Culture Collection, Manassas, Va., USA).
  • the EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule.
  • Tumor cells were grown as monolayer at 37° C. in a humidified atmosphere (5% CO2, 95% air).
  • the culture medium was RPMI 1640 containing 2 mM L-glutamine (ref: BE12-702F, Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (ref: 3302, Lonza).
  • EMT-6 tumor cells are adherent to plastic flasks.
  • tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (ref: BE02-007E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted and their viability was assessed by 0.25% trypan blue exclusion assay.
  • Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunocompetent rodents—R/M-H Extrudate, Sterile, filtrated at 0.2 ⁇ m water and Environmental enrichment (SIZZLE-dri kraft-D20004 SERLAB, France). Animals are individually identified with RFID transponder and each cage was ladled with a specific code. Treatment of the animals started after one week of acclimation for batches 2 and 3, or after three weeks of acclimation for batch 1.
  • mice On day ⁇ 14 (D-14), non-engrafted mice were randomized according to their individual body weight into 3 groups of 30 animals and 2 groups of 10 animals using Vivo Manager® software (Biosystemes, Couternon, France). The mice were separated into 3 batches of 10 animals per treatment group (batch 1: 10 animals of groups 1, 2 and 3; batch 2: 10 animals of groups 1, 2 and 3 and batch 3: 10 animals of groups 1 to 5) with different termination points from the start of the study: D-14 or D0.
  • batch 3 was split into 2 cohorts, due to termination and FACS analyses schedules; these were staggered over 1 day: D24/D25. Therefore, every cohort of animals had 5 animals per treatment group (4 animals from cage one and one animal from cage 2). Based on the ethical criteria, if the tumor volume were higher than 1500 mm 3 , the selection of the animals to be sacrifice on D24 and D25 is based on tumor volume instead of the cage.
  • the experimental design is depicted in FIG. 16A and summarized below:
  • mice of batches 2 and 3 were engrafted with EMT-6 tumour cells by a subcutaneous injection of 1 ⁇ 10 6 EMT-6 cells in 200 ⁇ L RPMI 1640 into the right flank (the 30 mice from batch 1, that were sacrificed on D14, did not receive the tumour injection).
  • the viability and behaviour of the animals was recorded every day. Body weights were measured twice a week. The length and width of the tumour was measured twice a week with callipers and the volume of the tumour was estimated by the following formula:
  • Tumour ⁇ ⁇ volume ⁇ Width 2 ⁇ Length 2
  • the treatment efficacy was assessed in terms of the effects of the test substance on the tumour volumes of treated animals relative to control animals.
  • the following evaluation criteria of antitumor efficacy were determined using Vivo Manager® software (Biosystemes, Couternon, France). Mean tumour volumes of groups 1 to 5 are depicted in FIG. 16B .
  • MRx0518+Anti-CTLA-4-treated group where a regression of tumour growth occurred from Day 14 post tumour induction.
  • MRx0518+Anti-CTLA-4 treatment significantly reduced tumour growth compared to the Vehicle-treated group on Day 21 and Day 24 post tumour induction.
  • the combination treatment of MRx0518 with Anti-CTLA-4 was the most efficacious for reducing tumour growth in BALB/c mice bearing subcutaneously grafted EMT6 tumours.
  • compositions comprising bacterial strain according to the invention tested the efficacy of compositions comprising bacterial strain according to the invention in a tumor model.
  • Test substance Bacterial strain #MRx0554.
  • Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131, isotype: Syrian Hamster IgG1, Bioxcell).
  • Test and reference substances vehicles Bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each day of injection to mice, antibody was diluted with PBS (ref: BE14-516F, Lonza, France).
  • Treatment doses Bacteria: 2 ⁇ 10 8 in 200 ⁇ L YCFA.
  • the anti-CTLA-4 was injected at 10 mg/kg/inj.
  • Anti-CTLA-4 was administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse weighing 20 g, 200 ⁇ L of test substance will be administered) according to the most recent body weight of mice.
  • Culture conditions of bacterial strain were as follows:
  • the EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule [71].
  • adherent tumor cells were detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.
  • BALB/C mice Healthy female BALB/C mice, of matching weight and age, were obtained from CHARLES RIVER (L′Arbresles) for the EMT6 model experiments.
  • Animals were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals were followed [72,73]. Animals were maintained in housing rooms under controlled environmental conditions: Temperature: 22 ⁇ 2° C., Humidity 55 ⁇ 10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation.
  • Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cm 2 cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, water from water bottles.
  • group housing 900 cm 2 cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, water from water bottles.
  • mice from the positive control group received anti-CTLA-4 antibody treatments.
  • Euthanasia Each mouse was euthanized when it reached a humane endpoint as described below, or after a maximum of 6 weeks post start of dosing.
  • Tumor ⁇ ⁇ volume ⁇ width 2 ⁇ length 2
  • Humane endpoints [75]: Signs of pain, suffering or distress: pain posture, pain face mask, behaviour; Tumor exceeding 10% of normal body weight, but non-exceeding 2000 mm 3 ; Tumors interfering with ambulation or nutrition; Ulcerated tumor or tissue erosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, emaciation, cachexia, dehydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia, significant bleeding; Neurologic signs: circling, convulsion, paralysis; Sustained decrease in body temperature; Abdominal distension.
  • Anaesthesia Isoflurane gas anesthesia was used for all procedures: surgery or tumor inoculation, i.v. injections, blood collection. Ketamine and Xylazine anesthesia was used for stereotaxia surgical procedure.
  • Analgesia Carprofen or multimodal carprofen/buprenorphine analgesia protocol were adapted to the severity of surgical procedure. Non-pharmacological care was provided for all painful procedures. Additionally, pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending veterinarian.
  • Euthanasia Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.
  • strain MRx0554 may be useful for treating or preventing other diseases associated with reduced immune system activity.
  • the Analytical Profile Index (API) test system consists of strips which contain miniaturised biochemical tests which assay for enzymatic activity in bacterial species. These tests are routinely used in the characterisation of novel strains.
  • API 50 CHL testing was carried out to examine carbohydrate metabolism in MRx0554. As per manufacturer's instructions, bacteria were cultured in 10 mL YCFA broth for 16-18 hours at 37° C. in an anaerobic workstation. This culture was diluted in 10 mL API CHL Medium so as to achieve a density roughly equivalent to McFarland standard No. 2, and 110 ⁇ l of this mixture was used to inoculate each cupule on a set of API 50 CH test strips. Test strips were incubated in a humidified incubation box at 37° C. in an anaerobic workstation for 48 hours, following which the colour of each cupule was recorded and assigned a value of negative, intermediate positive, positive or doubtful.
  • MRx0554 tested positive for fermentation of L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-saccharose (sucrose), D-trehalose, gentiobiose, D-tagatose, and potassium gluconate ( FIG. 18 ). Intermediate reactions were observed for D-mannitol, methyl ⁇ -D-glucopyranoside, D-lactose, D-raffinose, amidon (starch), and D-turanose.
  • HEK-BlueTM human TLR9 reporter cells (InvivoGen, San Diego, Calif., USA) were used to test the effect of MRx0518 on TLR9 activation.
  • HEK-BlueTM human TLR9 reporter cells (InvivoGen, San Diego, Calif., USA) were grown in DMEM supplemented with 10% (v/v) foetal bovine serum (FBS), 4 mM L-glutamine, 4.5 mg/mL glucose, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 100 ⁇ g/mL NormocinTM (InvivoGen), 10 ⁇ g/mL blastocidin (InvivoGen) and 100 ⁇ g/mL zeocin (InvivoGen) to 90% density. Cells were maintained at 37° C. and 5% CO2.
  • E. gallinarum MRx0518 was routinely cultured in Yeast extract, Casitone, Fatty Acid media (YCFA, E&O Laboratories, Bonnybridge, Scotland, UK) at 37° C. in an anaerobic cabinet (Don Whitley Scientific, Shipley, England, UK).
  • compositions were examined for their ability to induce TLR9 activation:
  • HEK-BlueTM human TLR9 reporter cells were incubated with the above treatments for 22 hours at 37° C. in a 5% CO2 atmosphere. Assays were developed using QUANTI-BlueTM (InvivoGen) as per manufacturer's recommendations for 2 h. The results depicted in FIG. 19 are an average from at least three independent experiments. Statistical significance was determined using the ordinary one-way ANOVA and Tukey's multiple comparisons tests.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • anti-CD3 Ebioscience, anti-CD3 monoclonal antibody (OKT3 clone), functional grade, cat. No. 16-0037-81
  • cRPMI contains RPMI 1640 (+L-Glut, 21875-034) 2 mM final conc.
  • the cells were incubated with viability dye (Viobility 405/520 Fixable Dye from Miltenyi biotec 1 ⁇ l/sample)+human Fc block, cat. 564219 (1 ⁇ l/sample) in PBS for 10 mins in the dark at room temperature.
  • viability dye Viobility 405/520 Fixable Dye from Miltenyi biotec 1 ⁇ l/sample
  • human Fc block cat. 564219 (1 ⁇ l/sample
  • the surface antibodies (2 ⁇ l of each) were then added directly to the wells for 10 mins in the dark at room temperature—CD3-APC-Vio 770 (Miltenyi, cat. No. 130-113-136), CD4-VioBlue (Miltenyi, cat. No. 130-114-534) and CD25-VioBright FITC (Miltenyi, cat. No. 130-113-283).
  • the cells were then washed twice in PBS and spun down at 300
  • the eBioscience FoxP3 transcription factor staining buffer was then used to fix and permeabilise the cells (cat. No. 00-5523). Following the eBioscience protocol, a perm/fix buffer was prepared using 1 part of concentrate solution and 3 parts of diluent. The cells were fixed for 1 h at RT and then washed 2 ⁇ in 1 ⁇ Perm wash and spun down at 300 g/5 min/RT.
  • Intracellular markers Transcription factors 2 ul anti-IL10-PE 5.5 ul anti-FoxP3- PE-Cy7 2 ul anti-IFN ⁇ -PE 5 ul anti-Tbet-APC Vio770 l0 ul anti-IL17a-APC 9 ul anti-RoRyt-PE
  • both supernatant of MRx0518 (SP 518) and heat-killed MRx0518 (HK 518) were able to induce differentiation of T helper cells and cytotoxic T cells, respectively, even in the absence of cytokines to induce differentiation (no cyto)
  • Splenocytes were isolated from C57BL/6 mice and plated in 96 well plates at a density of 900,000 cells/well in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep (Sigma-Aldrich) and 55 ⁇ M ⁇ -mercaptoethanol (Gibco). Cells were treated with different concentrations of blank media (YCFA + ) or bacteria supernatant from stationary phase for 72 hrs. Cell free supernatants were collected after each time point and stored at ⁇ 80° C. for cytokine analysis.
  • Cytokines were measured using multiplex procartaplex MO Th1/Th2/Th9/Th17/Th22/Treg 17plex kit (Invitrogen). Cell proliferation of untreated splenocytes or splenocytes treated by 10% YCFA medium or 10% MRx0518 bacteria supernatant was measured using MTT assay (Millipore), as depicted in FIG. 21F .
  • Live, growing MRx0518 bacteria were incubated for up to 2 h with the human intestinal epithelial cell line CaCo-2 and with the human monocyte/macrophage cell line THP-1.
  • the host response was analysed immediately (CaCo-2) or after a further 24 h incubation (THP-1).
  • Frozen healthy human PBMCs were purchased from Stem Cells Technologies (Cambridge UK). The cells were thawed and left to rest overnight in full growth media (RPMI 1640 with 10% FBS, 55 ⁇ M ⁇ -mercaptoethanol, 2 mM L. Glutamine and 100 U/mL penicillin, 100 ⁇ g/mL streptomycin) in CO 2 incubator at 37° C. For the experiment, cells were plated at a density of 750,000 cells/well in 48 well plates and treated in full growth media with 10% bacteria supernatants in the presence or absence of 1 ng/mL LPS. Cell culture media was added to untreated wells.
  • full growth media RPMI 1640 with 10% FBS, 55 ⁇ M ⁇ -mercaptoethanol, 2 mM L. Glutamine and 100 U/mL penicillin, 100 ⁇ g/mL streptomycin
  • FIGS. 21A-D Data are expressed in FIGS. 21A-D as an average of two technical replicates of 10 biological replicates (PBMC) or three biological replicates (splenocytes) and show production of cytokines in (A) PBMCs; (B) Splenocytes; (C) THP-1 cells; and (D) Caco-2 cells, following treatment with YCFA blank media (“Vehicle”) or MRx0518 bacteria/MRx0518 cell-free bacterial supernatant (“MRx0518”).
  • PBMC biological replicates
  • splenocytes splenocytes
  • NF- ⁇ B inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene with either the human NOD2 gene, TLR4, TLR9 or TLR5 genes (HEK-BlueTM-hNOD2, HEK-BlueTM-hTLR5, HEK-BlueTM-hTLR9 and FMK-BlueTM-hTLR4 cells, respectively, by InvivoGen, San Diego, Calif., USA).
  • SEAP embryonic alkaline phosphatase
  • HEK-TLR4 cells were maintained in DMEM 4.5 g/L D-glucose supplemented with 10% (v/v) heat-inactivated FBS, 4 mM L-Glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml normocin, 1 ⁇ HEK-Blue selection media; for HEK-TLR5 and HEK-TLR9 same media was used with the exception of the use of 2 mM L-Glutamine.
  • HEK-TLR5 and HEK-TLR9 were selected using 30 mg/ml and 10 mg/ml blasticidin respectively and 100 ⁇ g/ml zeocin media for both cell lines into the culture.
  • cells were washed with PBS, dissociated in PBS and collected in growth media.
  • Cells were plated in 96-well plates at a density of 25,000 cells/well for HEK-TLR4 and HEK-TLR5, 80,000 cells/well for HEK-TLR9 and 50,000 cells/well for HEK-NOD2.
  • the cells were treated with 1 ng/ml LPS (HEK-TLR4), 1 ng/ ⁇ l ultra-pure flagellin from Salmonella typhimurium (HEK-TLR5), 1 ⁇ M ODN2006 CPG (HEK-TLR9 positive control) or 1 ⁇ M ODN2006 GPC (HEK-TLR9 negative control), 1 ng/ml of L18-MDP and incubated in a CO 2 incubator at 37° C. Treatments proceeded for 22 h at 37° C.
  • LPS HEK-TLR4
  • HEK-TLR5 1 ng/ ⁇ l ultra-pure flagellin from Salmonella typhimurium
  • HEK-TLR9 positive control HEK-TLR9 positive control
  • 1 ⁇ M ODN2006 GPC HEK-TLR9 negative control
  • SEAP Secreted embryonic alkaline phosphatase
  • NF- ⁇ B promoter activation was measured in cells which were either untreated (“Untreated”), treated with YCFA+ medium (“YCFA”) or treated with MRx0518 (“MRx0518”).
  • the following positive controls (ing) were used—L18-MDP (for HEK-BlueTM-hNOD2 cells, FIG. 22A ), Lipopolysaccharide, LPS (for HEK-BlueTM-hTLR4, FIG. 22B ), CPG or negC (for HEK-BlueTM-hTLR9, FIG. 22C ) or recombinant flagellin from S.
  • FLA for HEK-BlueTM-hTLR5, FIG. 22D .
  • the cells were incubated with the various treatment at 37° C. in a 5% CO 2 atmosphere for 22 h.
  • QUANTI-BlueTM InvivoGen was mixed with cell supernatants, the plates were incubated for 2 h and optical density was measured at 655 nm.

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